With new ultramicro methodologies developed principally in our laboratory it should be possible to define the mineral formational agent for concentration of mineral ions in extracellular matrix vesicles using the micropuncture fluid and septa of growth cartilage in rachitic and healing rachitic rats. Evidence for the function of a mineral ion storage compound in matrix vesicles isolated by biochemical means and by ultracentrifugation of cartilage fluids will be studied with special reference to calcium-binding glycoproteins, nucleotides and alkaline phosphatase or neutral pyrophosphatases. Role of membrane-transport of mineral ions will also be studied; the role of lysozyme in inactivation of proteoglycans at calcifying sites will be further studied. Mg and pyrophosphate will be analyzed in isolated cartilage septa to determine their possible role as inhibitors of mineral ACP growth and conversion to HO apatite. If a glycoprotein storing calcium is found in the vesicle fractions, larger scale purification of pooled cartilages for the glycoprotein will be performed and antibodies to the glycoprotein will be labelled with ferritin, and presence of glycoprotein in various tissue sites will be determined by electron microscopy.